Localization of the large subunit of replication factor C near the 5 ' endof DNA primers

Replication factor C (RFC) is a heteropentameric sliding clamp loader prote in essential for processive synthesis of DNA by eukaryotic DNA polymerases delta and epsilon. To study the interaction of RFC with 3 ' and 5 ' ends of the DNA primer, we have developed chemical photocrosslinking assay using a synthetic DNA gap and DNA primer-template structures. We have found that t he radioactively labeled primers containing a photoreactive group at their 5 ' end could crosslink with the largest RFC subunit (RFC140) on primer-tem plates and DNA gap structures, but that 3 ' end photoreactive primers could only crossIink with RFC140 within the DNA gap structure. Addition of repli cation protein A (RPA) to the reaction mixture resulted in the crosslinking of RPA subunits and inhibited crosslinking of RFC140 using 3 ' but not 5 ' photoreactive primers present at the gap. The results suggest specific con tacts between RFC140 and the 5 ' end of the DNA primer. Together with previ ous data, these experiments allow us to propose a model for the DNA polymer ase switch during eukaryotic DNA replication. Copyright (C) 2001 John Wiley & Sons, Ltd.